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Abstract

Introduction

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and
proinflammatory processes. Abnormalities in these processes are primary features of
osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme
in heme degradation that confers cytoprotection against oxidative injury. Here, we
investigated the intracellular signaling pathways involved in thrombin-induced HO-1
expression in human synovial fibroblasts (SFs).

Methods

Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR.
The mechanisms of action of thrombin in different signaling pathways were studied
by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins
was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were
used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1
activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin,
and expression was higher than in normal SFs. OASFs stimulation with thrombin induced
concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors
or activators and genetic inhibition by siRNA of protease-activated receptors (PARs)
revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved
in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated
by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2).
Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.

Conclusion

Our results suggest that the interaction between thrombin and PAR1/PAR3 increases
HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling
pathways.

Introduction

Osteoarthritis (OA) is a degenerative disease characterized by a slow progressive
degeneration of articular cartilage, variable secondary synovial inflammation, and
osteophyte formation [1,2]. The exact etiology of OA is not well understood. In response to macrophage-derived
proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α
(TNF)-α, OA synovial fibroblasts (OASFs) produce chemokines that promote inflammation,
neovascularization, and cartilage degradation through activation of matrix-degrading
enzymes such as matrix metalloproteinases (MMPs) [3].

Heme oxygenase (HO)-1 is the key enzyme responsible for the degradation of heme to
carbon monoxide, free iron, and biliverdin-IX [4]. In mammals, biliverdin-IX is further converted to bilirubin-IX, an endogenous radical
scavenger with recently recognized antiinflammatory properties [5]. Free iron is rapidly sequestered into the iron-storage protein ferritin, leading
to additional antioxidant and antiapoptotic effects [6]. Carbon monoxide serves several biologic functions, with antiapoptotic and antiinflammatory
properties [7]. HO-1 is induced by various stimuli, such as lipopolysaccharide (LPS), proinflammatory
cytokines, and oxidants [8,9]. Recent findings revealed that HO-1 induction in animals protects them against the
development of arthritis [10]. HO-1 induction in OA chondrocytes results in decreased levels of MMPs and can exert
protective effects against cartilage degradation [11]. In synovial cells, HO-1 is an important factor regulating inflammation and cartilage
degradation during OA [12]. Therefore, HO-1 plays a crucial role in OA pathogenesis. However, the role of thrombin
in HO-1 expression in OA is still unknown.

Thrombin is a multifunctional enzyme that can activate hemostasis and coagulation
through the cleavage of fibrinogen to form fibrin clots. Tissue damage and fibrin
deposition are common features of inflamed synovium in OA, which indicates generation
of thrombin in the lesions and suggests an involvement of thrombin in the joint inflammation
[13]. Thrombin also acts as a mitogen to stimulate the abnormal proliferation of synovial
cells during rheumatoid arthritis (RA) and OA pathogenesis [14,15]. Thrombin activates intracellular signaling pathways by interacting with transmembrane
domain G protein-coupled receptors, known as protease-activated receptors (PARs).
PARs have been implicated in the development of acute and chronic inflammatory responses.
Three PARs, PAR-1, PAR-3, and PAR-4, are cleaved by thrombin, whereas PAR-2 is cleaved
by trypsin. It has been reported that thrombin induces HO-1 release in human microglia
[16], indicating that thrombin may play a role in the regulation of specific gene expression,
such as that of HO-1. However, the effect of thrombin on HO-1 expression in human
synovial fibroblasts has not yet been elucidated. In the present study, we explored
the intracellular signaling pathways involved in thrombin-induced HO-1 expression
in human OASFs. In our experiment, thrombin activated the PAR1/PAR3 receptor, PKCδ,
c-Src, and Nrf2 pathways, leading to the upregulation of HO-1 expression. These results
provide new insights into the mechanisms of thrombin action that may be of therapeutic
value for OA.

Cell cultures

We obtained approval from the local ethics committee, and subjects gave informed written
consent. Human synovial fibroblasts were isolated by collagenase treatment of synovial
tissue samples obtained from 32 patients with OA during knee-replacement surgeries
and 18 samples of nonarthritic synovial tissues obtained at arthroscopy after trauma/joint
derangement. The concentration of thrombin in the synovial fluid of selected patients
was measured with an enzyme-linked immunosorbent assay (ELISA) according to the protocol
provided by the manufacturer (Human thrombin ELISA kit; Abcam). OASFs were isolated,
cultured, and characterized, as previously described [17,18]. Experiments were performed by using cells from passages 3 to 6.

Quantitative real-time PCR

Total RNA was extracted from synovial fibroblasts by using a TRIzol kit (MDBio Inc.,
Taipei, Taiwan). The reverse-transcription reaction was performed by using 2 μg of
total RNA that was reverse transcribed into cDNA by using oligo (dT) primer [19,20]. The quantitative real-time PCR (qPCR) analysis was carried out by using Taqman one-step
PCR Master Mix (Applied Biosystems, Foster City, CA, USA). cDNA templates (2 μl) were
added per 25-μl reaction with sequence-specific primers and Taqman probes. Sequences
for all target gene primers and probes were purchased commercially (β-actin was used
as internal control) (Applied Biosystems). The qPCR assays were carried out in triplicate
on a StepOnePlus sequence-detection system. The cycling conditions involved 10-minute
polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 seconds and 60°C
for 60 seconds. The threshold was set above the nontemplate control background and
within the linear phase of the target gene amplification to calculate the cycle number
at which the transcript was detected (denoted CT).

Western blot analysis

Cellular lysates were prepared as described previously [21,22]. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride
(PVDF) membranes. The blots were blocked with 4% BSA for 1 hour at room temperature
and then probed with rabbit anti-human antibodies against PKCδ, c-Src, or Nrf2 (1:1,000)
for 1 hour at room temperature. After three washes, the blots were subsequently incubated
with donkey anti-rabbit peroxidase-conjugated secondary antibody (1:3,000) for 1 hour
at room temperature. The blots were visualized by enhanced chemiluminescence with
Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).

Transfection and reporter gene assay

Human synovial fibroblasts were co-transfected with 0.8 μg HO-1-luciferase plasmid
and 0.4 μg β-galactosidase expression vector. Fibroblasts were grown to 80% confluence
in 12-well plates and were transfected the following day with Lipofectamine 2000 (LF2000;
Invitrogen). DNA and LF2000 were premixed for 20 minutes and then applied to cells.
After 24-hour transfection, cells were incubated with the indicated agents. After
further 24-hour incubation, the media were removed, and cells were washed once with
cold PBS. To prepare lysates, 100 μl of reporter lysis buffer (Promega, Madison, WI,
USA) was added to each well, and cells were scraped from dishes. The supernatant was
collected after centrifugation at 13,000 rpm for 2 minutes. Aliquots of cell lysates
(20 μl) containing equal amounts of protein (20 to 30 μg) were placed into wells of
an opaque black 96-well microplate. An equal volume of luciferase substrate was added
to all samples, and luminescence was measured in a microplate luminometer. The value
of luciferase activity was normalized to transfection efficiency monitored by the
co-transfected β-galactosidase expression vector [23].

Kinase-activity assay

PKCδ activity was assessed with a PKCδ Kinase Activity Assay Kit according to manufacturer's
instructions (Assay Designs, MI, USA). The PKCδ activity kit is based on a solid-phase
ELISA that uses a specific synthetic peptide as a substrate for PKCδ and a polyclonal
antibody that recognizes the phosphorylated form of the substrate.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) analysis was performed as described previously
[24]. DNA immunoprecipitated by anti-Nrf2 antibody was purified. The DNA was then extracted
with phenol-chloroform. The purified DNA pellet was subjected to PCR. PCR products
were then resolved by using 1.5% agarose gel electrophoresis and visualized with UV.
The primers: 5'-CCATCAAACTTTAACTCGGTGA-3' and 5'- GACTTGGGAGATAGAAGGAACG-3' were used
to amplify across the human HO-1 promoter region (-857 to -752) [24].

Statistics

The values are reported as mean ± SEM. Statistical analysis between two samples was
performed by using the Student t test. Statistical comparisons of more than two groups were performed by using one-way
analysis of variance (ANOVA) with the Bonferroni post hoc test. In all cases, P < 0.05 was considered significant.

Results

Thrombin induces HO-1 expression in human synovial fibroblasts

It has been reported that clotting factors and fibrinolytic products are increased
in synovial fluid of patients with RA and OA [25]. Therefore, we examined the levels of thrombin expression in samples from patients
with OA and found that the expression of thrombin protein in human OASFs (Figure 1A, lines 4 to 6) was significantly higher than that in normal SFs (Figure 1A, lines 1 to 3). The OASFs medium showed a level of expression of thrombin that was
significantly higher than that seen in the medium from normal SFs (Figure 1B). In addition, concentrations of thrombin in synovial fluid were significantly higher
in patients with OA than in controls (Figure 1B). We applied thrombin directly to OASFs to examine the expression of HO-1. Treatment
of OASFs with thrombin (0.1 to 3 U/ml) for 24 hours induced HO-1 expression in a concentration-dependent
manner (Figure 1C, E), and this induction occurred in a time-dependent manner (Figure 1D, F). After thrombin (3 U/ml) treatment for 24 hours, the amount of HO-1 expression had
increased in OASFs (Figure 1D, F). To confirm further this stimulation-specific mediation by thrombin, PPACK, a thrombin
inhibitor, was used. Pretreatment of cells with PPACK effectively antagonized the
potentiating effect of thrombin on HO-1 expression (Figure 2A). These data indicated that thrombin increases HO-1 expression in human OASFs.

Figure 1.Thrombin induces HO-1 expression in human synovial fibroblasts. (A) Total protein was extracted from normal synovial fibroblasts (lines 1 and 2) and osteoarthritis
synovial fibroblast (OASF) cells (lines 3 to 6), and subjected to Western blot analysis
for thrombin. (B; left panel) Human synovial fibroblasts were cultured for 48 hours, and media were collected to
measure thrombin. (B; right panel) Synovial fluid was obtained from normal (n = 10) or osteoarthritis patients (n = 15) and examined with ELISA for the expression of thrombin. OASFs (six OA lines)
were incubated with various concentrations of thrombin for 24 hours (C, E) or with thrombin (3 U/ml) for 6, 12, or 24 hours (D, F). The HO-1 expression was examined with Western blotting and qPCR. Results are expressed
as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with the thrombin-treated group.

Involvement of PAR1/PAR3 receptor in thrombin-mediated increase of HO-1 expression

Thrombin exerts its effects through interaction with specific PAR1, PAR 3, and PAR4
receptors [26]. To investigate the role of PAR1, PAR3, and PAR4 subtype receptors in thrombin-mediated
increase of HO-1 expression, cells were treated with PAR1-, 3-, and 4-specific agonist
peptides and then examined for expression levels of HO-1. Of the agonist peptides
tested, the PAR1-selective receptor agonist peptide, SFLLRN-NH2 (100 μM) and TFRGAP-NH2 (PAR3 agonist peptide; 100 μM) significantly increased the expression of HO-1 (Figure 2A). In contrast, GYPGQV-NH2 (PAR4 agonist peptide; 100 μM) failed to upregulate HO-1 expression. Furthermore, thrombin, SFLLRN-NH2, and TFRGAP-NH2 only slightly increased HO-1 expression in normal synovial fibroblasts (Figure 2B), indicating that PAR agonist-induced HO-1 expression is more important during OA
pathogenesis. To confirm that PAR1 and PAR3 subtype receptors are involved in the
thrombin-mediated increase of HO-1 expression, specific inhibition of PAR-receptor
expression was accomplished with siRNA (Figure 2C). It was found that PAR1 and PAR3 receptor-specific siRNA but not PAR4-receptor siRNA
significantly blocked the thrombin-mediated increase of HO-1 expression (Figure 2D, E), indicating that interactions between thrombin and PAR1/PAR3 are important for HO-1
expression in human OASF cells.

The signaling pathways of PKCδ and c-Src are involved in the potentiating action of
thrombin

PKC has been shown to play an important role in cellular functions modulated by several
stimuli, including thrombin [27,28]. To determine whether PKC isoforms were involved in thrombin-triggered HO-1 expression,
OASFs were pretreated for 30 minutes with either GF109203X, a pan-PKC inhibitor, or
rottlerin, a selective PKCδ inhibitor [29], and then incubated with thrombin for 24 hours. As shown in Figure 3A and 3C, pretreatment with GF109203X and rottlerin but not PKCα inhibitor (Ro320432, 10 μM) reduced thrombin-induced HO-1 expression, which suggests that PKCδ plays a specific
role in thrombin-induced HO-1 expression in OASFs. Transfection of cells with PKCδ
siRNA also reduced thrombin-induced HO-1 expression (Figure 3B). We directly measured the PKCδ phosphorylation response to thrombin. Stimulation
of OASFs with thrombin led to a significant increase in phosphorylation of PKCδ (Figure
3D). The PKCδ activity in OASFs was increased by thrombin treatment in a dose-dependent
manner (Figure 3E). Pretreatment of cells with PPACK or transfection of cells with PAR1 and PAR3 siRNA
also reduced thrombin-mediated PKCδ kinase activity (Figure 3F). Based on these results, thrombin appears to act through a PAR1/PAR3 and PKCδ-dependent
signaling pathway to enhance HO-1 expression in human synovial fibroblasts.

Figure 3.PKCδ is involved in thrombin-induced HO-1 expression in synovial fibroblasts. (A, C) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30
minutes with GF109203X (3 μM), rottlerin (10 μM), and Ro320432 (10 μM) followed by stimulation with thrombin for 24 hours, and HO-1 expression was examined
with Western blotting and qPCR. (B) Cells (seven OA lines) were transfected for 24 hours with PKCδ siRNA followed by stimulation
with thrombin for 24 hours, and HO-1 expression was examined with qPCR. (C) Cells (six OA lines) were incubated with thrombin for indicated time intervals, and
PKCδ phosphorylation was examined with Western blotting. (D, E) Cells (seven OA lines) were incubated with thrombin for 30 minutes or pretreated for
30 minutes with PPACK or transfected 24 hours with PAR1 and PAR3 siRNA, followed by
stimulation with thrombin for 30 minutes, and PKCδ kinase activity was determined
with the PKCδ kinase kit. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

PKCδ-dependent c-Src activation is involved in the regulation of COX-2 expression
[30], and we investigated the role of Src in mediating thrombin-induced HO-1 expression
with the specific Src inhibitor PP2. As shown in Figure 4A and 4B, thrombin-induced HO-1 expression was markedly attenuated by pretreatment of cells
for 30 minutes with PP2 or by transfection of cells for 24 hours with c-Src mutant.
The major phosphorylation site of c-Src at the Tyr416 residue results in activation of c-Src autophosphorylation [31]. To examine directly the crucial role of c-Src in HO-1 expression, we measured the
level of c-Src phosphorylation at Tyr416 in response to thrombin. As shown in Figure 4C, treatment of OASFs with thrombin resulted in a time-dependent phosphorylation of
c-Src at Tyr416. Pretreatment of cells with PPACK and rottlerin markedly inhibited the thrombin-induced
c-Src kinase activity (Figure 4D). Based on these results, thrombin appears to act through a signaling pathway involving
the PAR1/PAR3 receptor, PKCδ, and c-Src to enhance HO-1 expression in human synovial
fibroblasts.

Figure 4.c-Src is involved in thrombin-induced HO-1 expression in synovial fibroblasts. (A, B) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30
minutes with PP2 (3 μM) or transfected with c-Src mutant for 24 hours followed by stimulation with thrombin
for 24 hours, and the HO-1 expression was examined with qPCR and Western blotting.
(C) OASFs (five OA lines) were incubated with thrombin for indicated time intervals, and
c-Src phosphorylation was examined with Western blotting. (D) Cells (six OA lines) were pretreated 30 minutes with PPACK or rottlerin, followed
by stimulation with thrombin for 30 minutes, and c-Src phosphorylation was examined
with Western blotting. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

Involvement of Nrf2 in thrombin-induced HO-1 expression

Activation of Nrf2 has been reported to play an important role in HO-1 expression
[32]. To determine the role of Nrf2 in thrombin-mediated HO-1 expression, OASFs were transfected
with Nrf2 siRNA. Transfection of cells with Nrf2 siRNA suppressed the thrombin-induced
HO-1 expression (Figure 5A). Stimulation of cells with thrombin also induced Nrf2 phosphorylation in a time-dependent
manner (Figure 5B). Pretreatment of cells with GF109203X, rottlerin, and PP2 attenuated thrombin-induced
Nrf2 activation (Figure 5C).

Figure 5.Thrombin induced Nrf2 activation in synovial fibroblasts. (A) Osteoarthritis synovial fibroblasts (OASFs; six OA lines) were transfected with Nrf2
siRNA for 24 hours, and HO-1 expression was examined with qPCR. (B) OASFs (six OA lines) were incubated with thrombin for indicated time intervals, and
Nrf2 phosphorylation was examined with Western blotting. (C) Cells (five OA lines) were pretreated 30 minutes with PPACK, rottlerin, or PP2 followed
by stimulation with thrombin for 30 minutes, and Nrf2 phosphorylation was examined
with Western blotting. Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group.

We next investigated whether Nrf2 binds to the ARE element on the HO-1 promoter after
thrombin stimulation. The in vivo recruitment of Nrf2 to the HO-1 promoter (-857 to -752) was assessed with chromatin
immunoprecipitation assays. In vivo binding of Nrf2 to the ARE element of the HO-1 promoter occurred after thrombin stimulation
(Figure 6A). The binding of Nrf2 to the ARE element by thrombin was attenuated by GF109203X,
rottlerin, and PP2 (Figure 6A).

Figure 6.PAR, PKCδ, and c-Src are involved in thrombin-induced Nrf2 activation. (A) Osteoarthritis synovial fibroblast (OASFs; seven OA lines) were pretreated with GF109203X,
rottlerin, and PP2 for 30 minutes followed by stimulation with thrombin for 120 minutes,
and ChIP assay was then performed. Chromatin was immunoprecipitated with anti-Nrf2
antibody. One percent of the precipitated chromatin was assayed to verify equal loading
(Input). OASFs (eight OA lines) were incubated with thrombin for 24 hours (B) or pretreated with PPACK, GF109203X, rottlerin, and PP2 for 30 minutes (C) or co-transfected with PAR1 siRNA, PAR3 siRNA, PAR4 siRNA, PKCδ siRNA, and c-Src mutant
for 24 hours (D) before incubation with thrombin for 24 hours. HO-1 luciferase activity was then assayed.
Results are expressed as the mean ± SEM. *P < 0.05 as compared with basal level. #P < 0.05 as compared with thrombin-treated group. (E) Schematic diagram of the signaling pathways involved in thrombin-induced HO-1 expression
in OASFs. Thrombin increases HO-1 expression by binding to the PAR1/PAR3 receptor
and activating PKCδ and c-Src, which enhances binding of Nrf2 to the ARE site. This
results in the transactivation of HO-1 expression.

To study further the pathways involved in the action of thrombin-induced HO-1 expression,
transient transfection was performed by using the human HO-1 promoter-luciferase construct.
Treatment of cells with thrombin led to dose-dependent increases in HO-1 promoter
activity (Figure 6B). PPACK, GF109203X, rottlerin, and PP2; PAR1, PAR3, and PKCδ siRNA; and c-Src mutant
all antagonized the stimulation of HO-1 promoter activity by thrombin (Figure 6C, D). These results indicated that thrombin-induced HO-1 expression is mediated through
the PAR1/PAR3, PKCδ, c-Src, and Nrf2 pathways in OASFs.

Discussion

OA is a heterogeneous group of conditions associated with defective integrity of articular
cartilage and related changes in the underlying bone. The chronic inflammatory process
is mediated through a complex cytokine network. It is not yet completely understood
which factors are responsible for initiating the degradation and loss of articular
tissues. It has been shown that thrombin acts as a mitogen to stimulate the abnormal
proliferation of synovial cells during RA and OA pathogenesis [14,15]. We hypothesized that thrombin is highly expressed during OA pathogenesis and promotes
the release of inflammatory cytokines as well as increased HO-1 production (an endogenous
compensation mechanism). First, we confirmed that synovial fluid concentrations of
thrombin were significantly higher in patients with OA than in normal fluid samples.
We then identified HO-1 as a target protein for the thrombin-signaling pathway in
OASF cells. Our results provided evidence that thrombin promotes the endogenous compensation
mechanism (that is, HO-1 expression), during OA pathogenesis. We found that thrombin
increased HO-1 production (the antiinflammatory response); however, this mechanism
is insufficient to arrest the progress of OA. Nonetheless, the discovery of the HO-1
signaling pathway may help us to develop effective therapy in the future.

Thrombin is known to activate three PARs, including PAR1, PAR3, and PAR4 [33]. However, we demonstrated that PAR1 and PAR3 but not PAR4 receptors were required
for thrombin-induced HO-1 production. Treatment with PAR1 or PAR3 agonist induced
HO-1 expression, but PAR4 agonist failed to upregulate HO-1 expression. Furthermore,
we could not inhibit thrombin-induced HO-1 upregulation by PAR4 receptor-specific
siRNA. These data suggest that PAR1 and PAR3 receptors are involved in thrombin-induced
HO-1 expression in human synovial fibroblasts.

Several isoforms of PKC have been characterized at the molecular level and have been
found to mediate a variety of cellular molecular responses [34]. We demonstrated that the PKC inhibitor GF109203X antagonizes the thrombin-mediated
potentiation of HO-1 expression, suggesting that PKC activation is an obligatory event
in thrombin-induced HO-1 expression in these cells. In addition, rottlerin but not
Ro320432 also inhibited thrombin-induced HO-1 expression.

One current report indicates that rottlerin is not a specific PKCδ inhibitor but inhibits
many other targets [35]. Therefore, we used PKCδ siRNA to confirm PKCδ function in OASFs. We found that PKCδ
siRNA inhibited the enhancement of HO-1 expression. Incubation of synovial fibroblasts
with thrombin also increased PKCδ phosphorylation and kinase activity. Conversely,
PPACK and PAR1 or PAR3 siRNA reduced thrombin-mediated PKC kinase activity. These
data suggest that the PAR1/PAR3 and PKCδ pathways are required for thrombin-induced
HO-1 expression.

Src, a tyrosine kinase, plays a critical role in the induction of chemokine transcription
[36]. Because c-Src is a downstream effector of PKCδ [30], we examined the potential role of c-Src in the signaling pathway of thrombin-induced
HO-1 expression. Treatment of cells with c-Src inhibitor PP2 or transfection of cells
with c-Src mutant reduced thrombin-mediated HO-1 expression. In addition, we found
that treatment of OASFs with thrombin promoted increases in c-Src phosphorylation.
These effects were inhibited by PPACK and rottlerin, indicating the involvement of
PKCδ-dependent c-Src activation in thrombin-mediated HO-1 induction.

Several binding sites exist for a number of transcription factors, including ARE in
the 5' promoter region of the HO-1 gene [37]. The results of this study show that Nrf2 activation contributes to thrombin-induced
HO-1 production in synovial fibroblasts, and that inhibition of the Nrf2-dependent
signaling pathway, including Nrf2 siRNA, inhibits thrombin-induced HO-1 expression.
We found that treatment of synovial fibroblasts with thrombin resulted in increased
Nrf2 phosphorylation. Furthermore, thrombin increased the binding of Nrf2 to the ARE
element within the HO-1 promoter, as shown by a chromatin immunoprecipitation assay.
Binding of Nrf2 to the ARE element was attenuated by GF109203X, rottlerin, and PP2.
By using transient transfection with HO-1-luciferase as an indicator of HO-1 activity,
we also found that thrombin induced an increase in HO-1 activity. In addition, PPACK,
GF109203X, rottlerin, and PP2 reduced thrombin-increased HO-1 promoter activity. Based
on these findings, we propose that the PAR1/PAR3, PKCδ, and c-Src pathways are involved
in thrombin-induced Nrf2 activation in human synovial fibroblasts.

Conclusions

We explored the signaling pathways involved in thrombin-induced HO-1 expression in
human synovial fibroblasts. We found that thrombin augmented HO-1 expression by binding
to the PAR1/PAR3 receptor and activating PKCδ and c-Src, which enhanced binding of
Nrf2 to the ARE site and resulted in HO-1 expression (Figure 6E). The discovery of this HO-1 signaling pathway helps us to understand the mechanism
of OA pathogenesis and may lead us to develop effective therapy in the future.

Competing interests

Authors' contributions

JFL performed the experiments. SMH, CYH, CHT, and WHY analyzed the data and provided
the suggestions. CTT conceived of and designed the experiments. All authors read and
approved the final manuscript.

Acknowledgements

This work was supported by grants from the National Science Council of Taiwan (NSC
100-2320-B-039-028-MY3) and Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-100-0401).
We thank Dr. S. Parsons for providing the c-Src mutant. We also thank the staff of
the Eighth Core Lab, Department of Medical Research, National Taiwan University Hospital
for technical support during the study.